U.S. patent number 6,610,652 [Application Number 10/095,654] was granted by the patent office on 2003-08-26 for microdose therapy.
This patent grant is currently assigned to Queen's University at Kingston. Invention is credited to Michael A. Adams, James D. Banting, Jeremy P. W. Heaton.
United States Patent |
6,610,652 |
Adams , et al. |
August 26, 2003 |
Microdose therapy
Abstract
Methods for treating vascular conditions associated with
localized imbalance in vascular tone, which are hypothesized to be
largely due to elevated endothelin (ET) are provided. The methods
involve administration of nitric oxide (NO), agents which are able
to provide NO, such as NO donors, agents which activate guanyl
cyclase, such as YC-1, or agents which prolong the actions of
endogenous NO or cyclic guanosine monophosphate (cGMP; a 2nd
messenger molecule), such as phosphodiesterase (PDE) inhibitors.
According to the invention, such agents are administered in minimal
doses or microdoses by any route known in the art, so as to provide
dosages which are about one half to about one twentieth (1/2 to
1/20) of those known to induce vasodilation in "normal"
circulations. The low doses of these agents effectively alleviate
vascular conditions associated with a reduction in NO production or
an attenuation of NO effect, by restoring balance in vascular tone
while exerting almost no systemic effect in normal vasculature.
Inventors: |
Adams; Michael A. (Kingston,
CA), Heaton; Jeremy P. W. (Gananoque, CA),
Banting; James D. (Kingston, CA) |
Assignee: |
Queen's University at Kingston
(Kingston, CA)
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Family
ID: |
26728335 |
Appl.
No.: |
10/095,654 |
Filed: |
March 8, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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613637 |
Jul 11, 2000 |
6423683 |
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469649 |
Dec 22, 1999 |
6165975 |
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PCTCA9800603 |
Jun 22, 1998 |
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Current U.S.
Class: |
424/718; 514/192;
514/565; 514/668; 514/470; 514/231.2; 514/13.5; 514/16.1;
514/10.2 |
Current CPC
Class: |
A61K
31/04 (20130101); A61K 38/063 (20130101); A61K
31/16 (20130101); A61P 15/00 (20180101); A61K
31/295 (20130101); A61P 15/10 (20180101); A61K
31/198 (20130101); A61P 43/00 (20180101); A61K
31/21 (20130101); A61P 15/02 (20180101); A61K
31/34 (20130101); A61K 31/5377 (20130101) |
Current International
Class: |
A61K
31/04 (20060101); A61K 31/16 (20060101); A61K
31/198 (20060101); A61K 31/28 (20060101); A61K
31/5377 (20060101); A61K 31/295 (20060101); A61K
31/185 (20060101); A61K 31/21 (20060101); A61K
38/06 (20060101); A61K 31/34 (20060101); A61K
31/5375 (20060101); A61K 038/00 (); A61K 031/43 ();
A61K 031/535 (); A61K 031/34 (); A61K 031/195 ();
A61K 031/13 () |
Field of
Search: |
;514/2,192,231.2,470,565,668 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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WO 95/19978 |
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Jul 1995 |
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WO |
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WO 96/32003 |
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Oct 1996 |
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WO |
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WO 96/38131 |
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Dec 1996 |
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WO |
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WO 97/03985 |
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Feb 1997 |
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WO |
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Other References
3M Delivery, "First CFC-free MDI Approved for Use in the United
States," Oct. 1996; vol. 8:1-11, plus 4 pages of Supplemental
Information provided herewith. .
Puckett, K.L., et al., "Alternate Packaging Technology for
Transdermal/Transmucosal Drug Delivery Systems," 3M Pharmaceuticals
Study (No Date Available). .
Wick. S.M., "Developing A Drug-in-Adhesive Design for Transdermal
Drug Delivery," Adhesives Age, Sep. 1995. .
Wolff, H., "Optimal process design for the manufacture of
transdermal drug delivery systems," PSTT, May 2000, vol. 3, No. 5;
pp. 173-181. .
Goldstein et al., "Oral Sildenafil in the Treatment of Erectile
Dysfunction," New England Journal of Medicine 1998
338(20):1397-1404. .
Webb et al., "Sildenafil Citrate and Blood-Pressure-Lowering Drugs:
Results of Drug Interaction studies with an Organic Nitrate and a
Calcium Antagonist," Am. J. Cardiology 1999 83(5A):21C-28C. .
Maurice et al., "Molecular Basis of the Synergistic Inhibition of
Platelet Function by Nitrovasodilators and Activators of Adenylate
Cyclase: Inhibition of Cyclic AMP Breakdown by Cyclic AMP,"
Molecular Pharm. 1990 37:671-681. .
Maurice et al., "Nitroprusside enhances isoprenaline-induced
increases in cAMP in rat aortic smooth muscle," Eur. J. Pharm. 1990
191:471-475..
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Primary Examiner: Henley, III; Raymond
Attorney, Agent or Firm: Townsend and Townsend and Crew
LLP
Parent Case Text
RELATED APPLICATIONS
This application is a continuation of U.S. patent application Ser.
No. 09/613,637, filed Jul. 11, 2000, now U.S. Pat. No. 6,423,683
which is Divisional Application of U.S. Application Ser. No.
09/469,649, filed Dec. 22, 1999, now U.S. Pat. No. 6,165,975, which
is a continuation of PCT/CA98/00603, filed Jun. 22, 1998, and
published as WO98/58633on Dec. 30, 1998, which application claims
the benefit of priority of U.S. Provisional Patent Application Nos.
60/050,491, filed Jun. 23, 1997, and 60/086,750, filed May 27,
1998, the contents of all these applications are incorporated
herein by reference in their entirety.
Claims
We claim:
1. A method for treating a urogenital disorder in a female subject,
said method comprising: administering to said female subject an
agent that enhances NO while maintaining normal systemic vascular
tone, to treat said urogenital disorder in said female subject.
2. The method for treating a urogenital disorder of claim 1,
wherein said urogenital disorder is selected from the group
consisting of decreased vaginal lubrication, decreased vaginal
engorgement, pain during intercourse, dyspareunia, urogenital
infections, effects on urogenitalia due to post-menopause or
diabetes, vascular disease, estrogen depletion conditions, sexual
dysfunction, and idiosyncratic vaginal dryness.
3. The method for treating a urogenital disorder of claim 2,
wherein said urogenital disorder is selected from the group
consisting of decreased vaginal engorgement, pain during
intercourse and dyspareunia.
4. The method for treating a urogenital disorder of claim 3,
wherein said urogenital disorder is dyspareunia.
5. The method for treating a urogenital disorder of claim 1,
wherein said administration of said agent is selected from the
group consisting of oral, sublingual, buccal, intravenous,
transdermal, vaginal, rectal, by inhalation, enteral, and
parental.
6. The method for treating a urogenital disorder of claim 1,
wherein administration of said agent is at concentration that is
about 1/2 to about 1/20 of a concentration of said agent required
to induce vasodilation in a healthy vasculature.
7. The method for treating a urogenital disorder of claim 6,
wherein administration of said agent is at concentration that is
about 1/4 to about 1/20 of a concentration of said agent required
to induce vasodilation in healthy vasculature.
8. The method for treating a urogenital disorder of claim 7,
wherein administration of said agent is at concentration that is
about 1/8 to about 1/16 of a concentration of said agent required
to induce vasodilation in healthy vasculature.
9. The method for treating a urogenital disorder of claim 1,
wherein said agent which enhances NO is GTN, and said level of
administration is a plasma concentration below about 250 pg/ml of
GTN.
10. The method for treating a urogenital disorder of claim 1,
wherein said agent is selected from the group consisting of NO, CO.
NO donors, CO donors, activators of guanylyl cyclase, PDE
inhibitors, and substances which produce an effect equivalent to
that of NO.
11. The method for treating a urogenital disorder of claim 10,
wherein said agent is a PDE inhibitor.
12. The method for treating a urogenital disorder of claim 10,
wherein said NO donor is selected from the group consisting of
glyceryl trinitrate, isosorbide 5-mononitrate, isosorbide
dinitrate, pentaerythritol tetranitrate, erythrityl tetranitrate,
sodium nitroprusside, 3-morpholinosydnonimine molsidomine,
S-nitroso-N-acetylpenicillamine, S-nitrosoglutathione, and
N-hydroxy-L-arginine.
13. The method for treating a urogenital disorder of claim 12,
wherein said NO donor is glyceryl trinitrate (GTN).
14. A method for improving blood flow to the vagina, said method
comprising: administering to a female subject an agent that
enhances NO while maintaining normal systemic vascular tone, to
improve blood flow to the vagina.
15. A method for treating dyspareunia in a female subject, said
method comprising: administering to said female subject an agent
that enhances NO while maintaining normal systemic vascular tone,
to treat dyspareunia in said female subject.
16. A method for treating a urogenital disorder in a female
subject, said method comprising: topically administering to said
female subject an agent that enhances NO while maintaining normal
systemic vascular tone, to treat said urogenital disorder in said
female subject.
17. A method for restoring NO/ET balance in a female subject, said
method comprising: administering to said female subject an agent
that enhances NO while maintaining normal systemic vascular tone,
to restore NO/ET balance in said female subject.
18. A method for treating an estrogen depletion condition in a
female subject, said method comprising: administering to said
female subject an agent that enhances NO while maintaining normal
systemic vascular tone, to treat said estrogen depletion condition
in said female subject.
19. A method for improving vaginal blood flow in a female subject,
said method comprising: administering to said female subject an
agent that enhances NO while maintaining normal systemic vascular
tone, to improve vaginal blood flow in said female subject.
20. A method for improving vaginal lubrication in a female subject,
said method comprising: administering to said female subject an
agent that enhances NO while maintaining normal systemic vascular
tone, to improve vaginal lubrication in said female subject.
Description
FIELD OF THE INVENTION
The field of invention is the treatment of conditions concerned
with peripheral vasoconstriction. More particularly, the invention
is concerned with methods for establishing normal vascular tone in
regions of the circulation which demonstrate pathophysiology. In
particular, the invention concerns a method of treating erectile
dysfunction by provision of nitric oxide, nitric oxide producing
agents, or activators of guanyl cyclase in small doses or
microdoses, i.e., doses that do not induce undesirable side
effects, such as systemic vasodilation, under normal
conditions.
BACKGROUND OF THE INVENTION
It is widely known that administration of nitric oxide (NO), or
compounds which deliver NO (i.e., NO donors, NO producing agents)
to a subject, can provoke powerful vasodilator responses. Such
administration is often accompanied by a number of undesirable side
effects which include headache, flushing, and hypotension.
The physiological role of NO has been described as that of a
powerful chronic vasodilator agent based on there being a marked
increase in vascular tone following NO synthase (NOS) blockade
(Johnson et al., Am. J. Hypertens. 5:919, 1992; Tolins et al.,
Hypertens. 17:909, 1991). The role of NO as a chronic vasodilator
has only been inferred by indirect means, i.e., by removal of NOS
activity. Endogenously, much more multiplicity and overlap in the
control of vasodilation can be inferred from the scientific
literature. For example, vasodilation can be induced by
acetylcholine, bradykinin, adenosine, adenosine triphosphate (ATP),
histamine, vasoactive intestinal polypeptide (VIP), and
leukotrienes, amongst others. The actions of these endogenous
modulators have been shown to be dependent, at least in part, on
the presence of the endothelium, an effect likely mediated by
endothelial derived relaxing factor/NO (EDRF/NO) (Garg, U. C. et
al., J. Biol. Chem. 266:9, 1991; Garg, U. C. et al., J. Clin.
Invest. 83:1774, 1989; Palmer, R. M. J. et al., Nature 327:524,
1987). Other vasodilator mechanisms exist which are not endothelium
dependent, such as .beta..sub.2 -adrenergic receptor activation,
atrial natriuretic peptide (ANP) and certain prostaglandins. The
actions of NO have been suggested to be mostly cGMP-mediated via
guanylate cyclase activation, although other mechanisms have been
suggested. For example, Garg et al. (J. Biol. Chem. 266:9, 1991; J.
Clin. Invest. 83:1774, 1989) and others (Assender, J. W. et al., J.
Cardiovasc. Pharmacol. 17(Suppl.3):S104, 1991; O'Conner, K. J. et
al., J. Cardiovasc. Pharmacol. 17(Suppl.3):S100-S103, 1991)
demonstrated a difference in the effects of NO-generating
vasodilator agents in inhibiting vascular smooth muscle cell growth
in culture; however, it is clear that NO can act not only as a
vasodilator but also to inhibit vascular growth responses in a
number of conditions (Farhy, R. D. et al., Circ. Res. 72:1202,
1993).
It has been believed and widely practised that NO, in humans and
animals, produced via sodium nitroprusside (SNP) infusion, causes
vasodilation in peripheral vasculature at doses greater than 10
.mu.g/kg per min. It has recently been determined that NO also
performs a function through interaction with endothelin (ET)
(Banting et al., J. Hypertens. 14:975, 1996; Richard et al.,
Circulation 91:771, 1995). Prior to this time, ET had been believed
to play a minimal role in maintaining tone in the peripheral
microvasculature and to have little impact on the state of
contraction of smooth muscle in those vessels. Recent studies have
indicated (Banting et al., J. Hypertens. 14:975, 1996) that ET is
under the inhibitory control of NO and that administration of NOS
inhibitors results in elevated levels of ET.
Endothelins were first described in 1988 and have been shown to be
powerful vasoconstrictors, predominantly found in vascular
endothelium and, since that time, numerous ET antagonists and their
pharmaceutically acceptable salts have been identified and can be
obtained commercially (e.g., Sigma, American Peptides). Detailed
descriptions of the chemical structures of various ET antagonists
may be found in U.S. Pat. No. 5,284,828 issued Feb. 8, 1994 to
Hemmi et al., U.S. Pat. No. 5,378,715 issued Jan. 3, 1995 to Stein
et al., and U.S. Pat. No. 5,382,569 issued Jan. 17, 1995 to Cody et
al. In addition, U.S. Pat. No. 5,338,726 issued Aug. 16, 1994 to
Shinosaki et al. describes the chemical structure of ET converting
enzyme inhibitors. To date, however, antagonists of ET have not
been approved for therapeutic use, although a number of
investigators have postulated that ET antagonists could be used for
conditions ranging from renal failure, endotoxic shock, asthma,
angina, or diabetes to pulmonary hypertension and possibly other
indications.
Under normal physiological conditions, ET can be found in almost
all parts of circulation at very low levels. In general, in the
normal rodent circulation ET is not found in elevated quantities
and appears to have little detectable role in the normal regulation
of vascular tone, i.e., there is no appreciable decrease in blood
pressure when an ET antagonist is administered by injection in
normal circulation. Further, at present there does not appear to be
any evidence suggesting that ET plays a physiological role even in
a small portion of the circulation under normal circulatory
conditions in experimental models. However, it is likely that the
systemic circulation may appear to be normal when, in fact,
specific regions of the circulation are undergoing
pathophysiological changes such as occurs in conditions such as
erectile dysfunction (ED) (Adams et al., Int. J. Impot. Res.
9:85-91, 1997).
Consequently, there are cardiovascular conditions which are
traditionally treated in human beings by significant doses of NO or
NO donors, such as glyceryl trinitrate (GTN) (0.2 mg/h and
greater). However, these doses are known to induce systemic
vasodilation and provoke considerable overall systemic side effects
(The, L. S. et al., Brit. J. Rheum. 34:636, 1995). This is
particularly so where a pathological condition exists only in
certain major organs (e.g., heart, kidney, liver). As a result, a
satisfactory method for promoting recovery of normal perfusion
pressure in organs with certain pathologies without producing
overall systemic hypotension has not been discovered.
Based on the understanding that a significant portion of underlying
problems in clinical erectile dysfunction relates to "vascular"
mechanisms, much of the current state-of-the-art research involves
determining the contribution that the different vascular effector
control systems make in normal and pathophysiological states. There
is substantial understanding of hemodynamic events that lead to an
erection, and yet the quantitative roles of each of the
neuroeffector, humoral and local systems in these events remain
poorly described. Since 1990, NO has been considered the primary
non-adrenergic non-cholinergic neurotransmitter in the penis and
has been presumed to be the primary mediator of corporal relaxation
during erection (Ignarro L. J. et al., Biochem. Biophys. Res.
Comm., 170:843, 1990).
It is well established that, for an erection to occur, neurally
mediated (autonomic) vasodilation of the penile arterial blood
vessel and the trabecular meshwork takes place (Lue, T. F. et al.,
J. Urol. 137(5):829, 1987) permitting increased blood flow into the
cavernous bodies of the penis. The expanding intra corporal volume
compresses the effluent veins that lie between the erectile tissue
and the surrounding fibrous, relatively inelastic, tunica
albuginea. The outflow capacity is thereby decreased and entrapment
of blood ensues, resulting in the transformation of the flaccid
penis into its erect state (Lue, T. F. et al, J. Urol. 137(5):829,
1987; Juenemann, K. P. et al., J. Urol. 136(1):158, 1986; Lue, T.
F. et al., J. Urol. 130:1237, 1983; Weiss, H. et al., Ann Intern.
Med. 76:793, 1980). The level of arterial vascular tone (i.e.,
blood pressure) is one of critical importance in this process,
although adequate perfusion pressure is also a necessary factor.
The converse, detumescence, is mediated by the sympathetic nervous
system (Saenz de Tajada, I. et al., Am. J. Physiol. 254:H459, 1988;
Juenemann, K. P. et al., Br.J. Urol. 64, 1989).
The issue of "impotence" (defined as "a pattern of persistent or
recurrent inability to develop or maintain an erection of
sufficient rigidity for successful coitus") was discussed at a
consensus conference of the National Institutes of Health (NIH) in
Washington in December 1992 and has been clearly identified as
having a wide range of causative or associated factors. The
Massachusetts Male Aging Study (MMAS) has provided an updated view
of the epidemiology of erectile dysfunction. It is accepted that
the prevalence of impotence increases with age (Kinsey A. C. et
al., "Sexual Behaviour in the Human Male", W. B. Saunders:
Philadelphia, 1948). Severe or complete ED increases from 5 to 15%
between 40 and 70 years of age, (Feldman, H. A. et al., J. Urol.
151:54, 1994). ED has been shown to be "directly correlated with
heart disease, hypertension, diabetes, associated medications,
indices of anger and depression, and inversely with serum
dehydroepiandrosterone, high density lipoprotein, cholesterol and
an index of dominant personality."
It is now estimated that in North America there are more than
30,000,000 men with some form of ED, a significant increase from
the figure of 10,000,000 quoted just 10 years ago (Shabsigh, R. et
al., Urology 32:83, 1988; Whitehead E., Geriatrics 43(2):114, 1988;
Furlow, W. L. et al., Med. Aspects Human Sexuality 19:13, 1985).
From these figures it is also reasonable to estimate that as many
as three million Canadian men may have a degree of ED. The direct
cost of treating impotence is significant. Reliable figures from
1985 show that the cost of treating impotence exceeded 146 million
dollars in the United States in that year alone (National Center
for Health Statistics) and this number is just the estimated market
size for one type of injectable therapy. The secondary effects and
indirect costs associated with ED suggest that impotence and sexual
dysfunction are medical icebergs. The consequences of sexual
dysfunction may be seen in strains on the host relationship
potentially leading to marital breakdown, violence, work related
sequelae, deviant sexual behaviour and impacts on children, when
present, that can carry the damage into a new generation of
unwanted behaviours. If ED underlies even a small but significant
percentage of marital and family breakdown, then it adds vastly to
the social and economic burden in society. The pragmatic issue is
that large numbers of men are now being treated for ED and most of
the treatments are fairly blunt instruments (e.g., intracavernosal
injection (ICI)) of mixed vasoactive compounds, penile prosthesis
insertion) with significant cost and complications (ICI: pain,
priapism, dislike of the technique; prostheses: reoperation,
infection, distortion of body image).
Administration of NO, or compounds which are able to deliver NO,
have been suggested as possible therapies; however, these agents
can provoke powerful yet inappropriate vasodilator responses (Brock
et al., J. Urol. 150:864, 1993). Such administration is often
accompanied by a number of undesirable side effects related to
systemic vasodilation which include headache, flushing, and
hypotension. Consequently, there is a real need to provide methods
whereby ED and other vascular disease may be quickly and
effectively treated without any inappropriate side effects.
SUMMARY OF THE INVENTION
Problems associated with localized imbalance in vascular tone, such
as seen in ED, and which are hypothesized to be largely due to
elevated ET, may be relieved by the administration of agents which
are able to provide NO, by direct administration of NO, or by
administration of an agent or agents which activates guanyl
cyclase, such as, for example, YC-1, or other agents which prolong
the actions of endogenous NO or of cGMP (a 2nd messenger molecule),
such as phosphodiesterase (PDE) inhibitors, in minimal doses or
microdoses, which heretofore had not been thought to result in
effective treatment of an imbalance in vascular tone.
In the normal physiological state, there are sufficient quantities
of NO present in the vasculature to maintain appropriate levels of
ET (Banting et al., J. Hypertens. 14:975, 1996). The addition of
further NO has little impact on the effect of ET and consequently
any further vasodilation seen in such normal smooth muscle in the
vasculature is likely attributable to NO's effects on the
generation of cGMP, with cGMP resulting in decreased levels of
Ca.sup.++. In certain pathological conditions, such as diabetes and
cardiovascular disease, and/or as a consequence of age, tissue is
unable to provide satisfactory levels of NO in order to suppress
normal levels of ET present in smooth muscle tissues.
As such, physiological conditions where NO production is reduced in
a specific local circulation, such as male ED, indicate that
suppression of ET activity should offer an effective treatment.
Consequently, in accordance with this, the present invention
provides for the use of an agent or agents which directly or
indirectly generates NO at dosages which are about one half to
about one twentieth (1/2 to 1/20) of those known to induce
vasodilation in "normal" circulations, and consequently exert
almost no effect in the normal vasculature. As such these low
doses, or "microdoses" are hypothesized to normalize the balance
between NO and ET. The range of about 1/2 to about 1/20 is derived
from the observation that at doses which are below about 1/2 the
normal dose, systemic effects are generally no longer seen. At
about 1/20 the normal dose, however, the desired effect is also
generally no longer observed, i.e., there is no effect.
According to one aspect of the present invention the concept of
"low-dose" agents which directly or indirectly generate NO, or
prolong the action of NO, or enhance the cGMP 2nd messenger system,
such as PDE inhibitors, is also applicable to any other peripheral
pathological conditions where, regardless of the origin, NO is at
least partially inhibited.
According to a further aspect of the present invention, there is
provided a method for restoring normal vascular tone in vasculature
where NO levels are depleted and restoration of such levels may be
achieved by addition of NO at levels which do not appreciably alter
normal systemic vascular tone.
According to yet a further aspect of the present invention there is
provided a method to treat any condition where NO levels are at
least partially inhibited or reduced, wherein the method comprises
administration of NO or one or more NO donors, or one or more
agents which activate guanyl cyclase, by oral, sublingual, buccal,
intravenous, transdermal, or any other effective route, in
concentrations that are about 1/2 to about 1/20 of a concentration
required to induce vasodilation in "healthy" or normal regions such
as the coronary or skeletal muscle vasculature. Preferably, the
concentrations of the method of the present invention are about one
quarter to about one twentieth (1/4 to 1/20) of a concentration
required to induce vasodilation in "healthy" or normal regions such
as the coronary or skeletal muscle vasculature. Still more
preferably, the concentrations of the method of the present
invention are about one eighth to about one sixteenth (1/8 to 1/16)
of a concentration required to induce vasodilation in "healthy" or
normal regions such as the coronary or skeletal muscle
vasculature.
According to one aspect of the present invention there is provided
a method to treat instances of renal disease associated with
excessive vasoconstriction where NO levels are at least partially
inhibited, wherein the method comprises administration of NO or one
or more NO donors, or one or more agents which activate guanyl
cyclase, by oral, sublingual, buccal, intravenous, transdermal, or
any other effective route, in concentrations that are about 1/2 to
about 1/20 of a concentration required to induce vasodilation in
"healthy" or normal regions of the circulation such as the coronary
or skeletal muscle vasculature.
According to a another aspect of the present invention there is
provided a method to treat premature aging of the skin associated
with inappropriate vasoconstriction of the skin vasculature which
is associated with at least partial inhibition of NO levels in the
skin, wherein the method comprises administration of NO or one or
more NO donors, or one or more agents which activate guanyl
cyclase, by oral, sublingual, buccal, intravenous, transdermal, or
any other effective route, in concentrations that are about 1/2 to
about 1/20 of a concentration required to induce vasodilation in
"healthy" or normal regions such as the coronary or skeletal muscle
vasculature.
According to a further aspect of the present invention there is
provided a method to treat male ED caused at least by partial
inhibition of NO in the penile vasculature wherein the method
comprises administration of NO or one or more No donors, or one or
more agents which activate guanyl cyclase, by oral, sublingual,
buccal, intravenous, transdermal, or any other effective route, in
concentrations that are about 1/2 to about 1/20 of a concentration
required to induce vasodilation in "healthy" or normal regions such
as the coronary or skeletal muscle vasculature.
According to yet a further aspect of the present invention there is
provided a method to treat ED comprising administering to a subject
in need thereof a quantity of glyceryl trinitrate (GTN) by any
route, for example, oral, sublingual, transdermal, intravenous, or
inhalation, that provides plasma concentrations of below about 250
pg/ml of the GTN, so that ED is treated.
According to one aspect of the present invention there is provided
a low-dose transdermal "patch" with short term release of, for
example, GTN or any other effective provider of NO, such as, for
example, one or more NO donors or one or more agents which activate
guanyl cyclase, over a period of time less than 6 hours (as opposed
to 12, 18 or 24 hour release), which restores normal vascular tone
in an affected local vascular bed, such as the pudendal or penile
vasculature of men with ED, without inappropriately affecting
systemic vascular tone or causing hypotension, and preferably, but
not necessarily, without inducing tolerance (Bennett et al., Circ.
Res. 63:693, 1988) to the effects and/or biotransformation of a NO
releasing compound to its releasing form.
According to a further aspect of the present invention, there is
provided a low dose or microdose "patch" with long term release of,
for example, GTN, or any other effective provider of NO, such as,
for example, one or more NO donors, or one or more agents which
activate guanyl cyclase, over a period of time greater than 6 hours
(typically 12 to 24 hour release), which restores normal vascular
tone in an affected local vascular bed, such as the pudendal or
penile vasculature of men with ED, without inappropriately
affecting systemic vascular tone, and preferably, but not
necessarily, without inducing tolerance to the effects of a NO
releasing compound and/or biotransformation of a NO releasing
compound to its releasing form.
According to a further aspect of the present invention there is
provided a method to treat female sexual dysfunction (SD) wherein
the method comprises administration of NO or one or more NO donors,
or one or more agents which activate guanyl cyclase, by oral,
sublingual, buccal, intravenous, transdermal, or any other
effective route, in concentrations that are about 1/2 to about 1/20
of a concentration required to induce vasodilation in "healthy" or
normal regions such as the coronary or skeletal muscle
vasculature.
According to yet a further aspect of the present invention there is
provided a method to treat SD comprising administering to a subject
in need thereof a quantity of GTN by any route, for example, oral,
sublingual, transdermal, intravenous, or inhalation, that provides
plasma concentrations of below about 250 pg/ml of the GTN, so that
SD is treated.
According to one aspect of the present invention there is provided
a low-dose transdermal "patch" with short term release of, for
example, GTN, or any other effective provider of NO such as, for
example, one or more NO donors or one or more agents which activate
guanyl cyclase, over a period of time less than 6 hours (as opposed
to 12, 18 or 24 hour release), which restores normal vascular tone
in an affected local vascular bed, such as the pudendal, cervical
or vaginal vasculature of women with SD, without inappropriately
affecting systemic vascular tone or causing hypotension, and
preferably, but not necessarily, without inducing tolerance
(Bennett et al., Circ. Res. 63:693, 1988) to the effects and/or
biotransformation of a NO releasing compound to its releasing
form.
According to a further aspect of the present invention there is
provided a low dose or microdose "patch" with long term release of,
for example, GTN, or any other effective provider of NO, such as,
for example, one or more NO donors or one or more agents which
activate guanyl cyclase, over a period of time greater than 6 hours
(typically 12 to 30 hour release), which restores normal vascular
tone in an affected local vascular bed such as the pudendal,
cervical or vaginal vasculature of women with SD without
inappropriately affecting systemic vascular tone, nor inducing
tolerance to the effects of a NO releasing compound and/or
biotransformation of a NO releasing compound to its releasing
form.
Definitions GTN glyceryl trinitrate ISMN isosorbide 5-mononitrate
ISDN isosorbide dinitrate PETN pentaerythritol tetranitrate ETN
erythrityl tetranitrate SNP sodium nitroprusside SIN-1
3-morpholinosydnonimine molsidomine SNAP
S-nitroso-N-acetylpenicillamine SNOG S-nitrosoglutathione NOHA
N-hydroxy-L-arginine cAMP cyclic adenosine monophosphate cGMP
cyclic guanosine monophosphate L-NAME N.sup..omega.
nitro-L-arginine methyl ester IP.sub.3 inositol-1,4,5-triphosphate
RIHP renal interstitial hydrostatic pressure T tumescence R
rigidity
"Applying various forms of NO" as used herein includes
administering NO donor or NO producing agents.
"Enhancing penile erection" as used herein is understood to mean
increasing physical size and improving tumescence and/or rigidity
of a penis, preferably so that it is capable of intromission.
"Erection of good quality" and "effective erection" are used herein
interchangeably to mean adequate for vaginal penetration (i.e.,
intromission, or intercourse).
"NO donor", "NO producing agent", "NO delivering compound", "NO
generating agent" and "NO provider" are used interchangeably in
this specification and include all compounds which donate NO
through biotransformation, compounds which generate NO
spontaneously, compounds which spontaneously release NO, or any
other compounds which otherwise generate NO or an NO-like moiety
and include: glyceryl trinitrate, isosorbide 5-mononitrate,
isosorbide dinitrate, pentaerythritol tetranitrate, erythrityl
tetranitrate, sodium nitroprusside, 3-morpholinosydnonimine
molsidomine, S-nitroso-N-acetylpenicillamine, S-nitrosoglutathione,
N-hydroxy-L-arginine, S,S-dinitrosodithiol, or NO gas, or a
functional equivalents thereof. In some cases, NO is generated by
activation of guanyl cyclase.
"Penis" as used herein may be interpreted to apply equally to
clitoris in so far as there is substantial equivalence between
penile and clitoral erectile tissue. "Sexual dysfunction" (SD) as
used herein includes aspects of female dysfunction and urogenital
aging such as decreased vaginal lubrication, decreased vaginal
engorgement, pain during intercourse such as, for example,
dyspareunia, urogenital infections; and urogenitalia as affected by
post-menopause, diabetes, vascular disease, estrogen depletion
conditions, sexual dysfunction, and idiosyncratic vaginal dryness,
respectively.
"Various forms of NO" as used herein is understood to mean any one
of NO.sup..multidot., NO.sup.+ and NO.sup.-, preferably NO.sup.+
and NO.sup.-, and can include as an alternative CO (carbon
monoxide) in its various forms, which produces an equivalent effect
to NO.
"Without inappropriately affecting systemic vascular tone" as used
herein means not affecting mean arterial pressure so as to produce
inappropriate systemic vasodilation with effects such as
hypotension, headache, flushing.
BRIEF DESCRIPTION OF THE DRAWINGS
Embodiments of the invention will now be described, by way of
example, with reference to the accompanying drawings, wherein:
FIG. 1 is a graphical representation of results from measurements
of mean arterial blood pressure in rats and illustrates the
differential impact on mean arterial pressure (mmHg) of infusions
of sodium nitroprusside in control and L-NAME treated rats.
FIG. 2 is a representative tracing of measured mean arterial
pressure in a rat demonstrating the contrasting effect of low level
infusions of sodium nitroprusside in control and acute L-NAME
treated rats.
FIG. 3 is a graphical representation of measurements of mean
arterial blood pressure illustrating an enhanced glyceryl
trinitrate dose response curve in rats treated with control and
L-NAME, acutely.
FIG. 4 is a schematic diagram depicting a range representative of a
"normal" vascular controlling "window" of NO with comparisons of
control and L-NAME treated rats.
FIG. 5 is a representative tracing of changes in mean arterial
pressure and renal interstitial hydrostatic pressure (RIHP) in
respect of renal circulatory blockade of NO synthase.
FIG. 6 is a representative tracing of changes in mean arterial
pressure and renal interstitial hydrostatic pressure (RIHP) in
respect of the capacity of L-arginine to induce decreases in RIHP
in connection with the renal circulation.
FIG. 7 is a representative tracing of changes in mean arterial
pressure and renal interstitial hydrostatic pressure (RIHP) in
respect of decreasing RIHP following removal of L-arginine in the
renal circulation.
FIG. 8 is a graph comparing the sensitivity of mean arterial
pressure to the NO donor sodium nitroprusside (SNP, right) and to
isopropylnorepinephrine (INE, left), in the presence and absence of
the NO synthase blocker L-NAME.
FIG. 9 is a cumulative dose response curve of mean arterial
pressure to prazosin, an .alpha..sub.1 blocker, in the presence and
absence of NO synthase blockade (L-NAME).
FIG. 10 is a graph illustrating a change in mean arterial pressure
from control conditions in the presence of SNP low-dose,
SNP+L-NAME, and L-NAME alone.
FIGS. 11 and 12 are graphs illustrating a dose-response
relationship of mean arterial pressure for different doses of the
nitric oxide donors SNP (top panel) and GTN (bottom panel), before
and after both nitric oxide synthase blockade (L-NAME) and
endothelin receptor antagonism (PD145065).
DETAILED DESCRIPTION OF THE INVENTION
The physiological role of NO has been described as that of a
chronic vasodilator agent acting to persistently "offset" the
actions of a number of vasoconstriction systems. However, as
discussed by Banting et al. (J. Hypertens. 14:975, 1996), based on
this rationale, this would reveal that the NO vasodilator system
would normally have an overall activity level at close to 70% of
the NO system's maximal capacity, leaving the system little reserve
or gain to protect against further deviations in mean arterial
pressure and blood flow homeostasis.
Banting et al. (J. Hypertens. 14:975, 1996) proposed that the
chronic role of NO, in vivo, is not that of a chronic vasodilator
system but rather an inhibitor of the activity of local
vasoconstrictor agents, such as ET. That is, the hypertension
following blockade of NO production was completely reversed with
the administration of an ET receptor antagonist. This finding,
combined with the understanding that ET appears to play almost no
role in the "normal" physiological control of resting mean arterial
pressure, indicates that the function of NO may be different than
previously proposed. The results of the Quantitative Study as
detailed below, in summary, indicate that: (i) the amount of NO
required to completely restore mean arterial pressure in L-NAME
treated rats from hypertensive levels is much less than that
required to lower mean arterial pressure under control conditions;
(ii) the increase in sensitivity to NO and NO donors allows for as
little as 1/20th of the standard concentration to provide a given
vasodilator response in NO synthase blockade rats; (iii) the level
of NO required to completely reverse the hypertension in the
chronic phase of L-NAME induced hypertension without altering blood
pressure in control rats is at the same level as required to
reverse the hypertension following acute NO synthase blockade; and
(iv) the similar mean arterial pressure lowering, in both treated
and control rats, that occurred with the administration of "high"
concentrations of sodium nitroprusside suggests that signalling
processes have been "normalized" at approximately 10-12 ug/kg per
minute of sodium nitroprusside infusion.
Banting et al. (J. Hypertens. 14:975, 1996) have suggested that a
major role of NO is not to function as a chronic vasodilator, but
rather a chronic inhibitory regulator of ET-mediated
vasoconstriction. In that study it was demonstrated that
ET-mediated vasoconstriction does not contribute to the maintenance
of vascular tone in a circulation with an intact NO generating
system. Also, it was demonstrated that almost the entire L-NAME
induced hypertension was both prevented and/or reversed via the
administration of an ET receptor antagonist. The results detailed
below under the heading Quantitative Study quantitate the exogenous
level of NO required to restore this regulatory balance and, taken
together, we propose that NO functions to suppress ET-mediated
vasoconstriction within a physiological "window" as illustrated in
FIG. 4, in a manner which is unrelated to its direct role as a
vasodilator substance. Indeed, it is proposed that the mechanism of
action of the NO donors delivered at the micro doses disclosed
herein, are not vasodilatory; rather they act to suppress the
endogenous role of endothelin.
These conclusions and their applicability to other
microcirculations are supported by studies conducted in the kidney
in connection with measurements of renal interstitial hydrostatic
pressure and mean arterial pressure as reported below under the
heading: Effect of NO in Renal Vasculature.
In a further application of the present invention, studies
conducted in human males with a low-dose "patch" with up to 3 hours
of release of effective NO, demonstrate that providing a
"microdose" of NO restored normal erectile function. The results of
these studies are detailed below under the heading: Clinical Data:
For erectile dysfunction reversal with microdoses of NO.
Accordingly, the present invention provides a method for restoring
normal vascular tone through the use of an agent or agents which
directly or indirectly generate(s) NO at dosages of about 1/2 to
about 1/20 of those currently used in clinical applications.
The method of the present invention is also applicable to any other
peripheral pathological conditions where regardless of the origin,
NO is at least partially inhibited. In respect of the conditions
detailed below evidence in the literature demonstrates that the
production of NO is, at least partially, inhibited. This leads to
an imbalance between NO and ET in favour of ET. The method of the
present invention restores this balance with `quantities` of NO
that do not induce vasodilation, or not appreciably, in `healthy`
or non-NO deficient circulations. Conditions wherein the present
invention has application include any condition where regional
circulation exhibits inappropriate vasoconstriction, such as, for
example, ED, conditions associated with female sexual dysfunction
involving vaginal and/or pelvic circulation, Raynaud's phenomenon
(a condition of the fingers with constrained blood flow), as well
as conditions associated with inappropriate vasoconstriction in,
for example, cutaneous and/or dermal (for example in reconstructive
surgery, as well as scleroderma and diffuse cutaneous systemic
sclerosis), cerebral, and renal circulations. Concerning ED, the
therapy of the present invention can be used to restore "normal"
vasculature response in a patient who has undergone a radical
prostatectomy procedure where normal circulatory control may not
return for 6-8 months post operatively.
The therapy of the present invention therefore has application in
female dysfunction and urogenital aging conditions such as vaginal
lubrication, vaginal engorgement, pain during intercourse
(dyspareunia), urogenital infections; and urogenitalia as affected
by post-menopause, diabetes, vascular disease, estrogen depletion
conditions, sexual dysfunction, and idiosyncratic vaginal dryness,
respectively. In such situations, the therapy of the present
invention works to improve blood flow to the vagina leading to
vaginal engorgement promoting better circulation (lubrication).
It is understood that any agent which provides a dose range of NO
is intended to be included within the scope of the present
invention. Thus, in accordance with the invention, an agent may be
NO, an NO-like substance, a substance which directly releases NO
(e.g., an NO donor) or causes the release of NO, a substance having
NO-like activity or effect, a substance which directly activates
guanyl cyclase, or a substance that prolongs the actions of the 2nd
messenger molecule cGMP, such as a PDE inhibitor. Regardless of
which of such agent or agents is employed in the methods of the
invention, what is important is that an enhanced level of NO is
achieved, or the effect of an enhanced level of NO is achieved. The
enhanced level is delivered to the vasculature such that normal
vascular tone is achieved by a mechanism which involves normalizing
levels of ET, thereby restoring the tone of the target
microvasculature.
It should also be understood that the methods of the invention
include the administration of an agent or agents as described
above, (i.e, NO, an NO-like substance, a substance which directly
releases NO (e.g., an NO donor) or causes the release of NO, a
substance having NO-like activity or effect, or a substance which
directly activates guanyl cyclase), either acutely or chronically.
Acute administration is administration of a finite duration and can
be, for example, as short as that associated with treatment of ED,
wherein administration takes place for a prescribed period of time
(e.g., up to several hours) at discrete instances when the affect
of the administration (penile erection) is desired. Acute
administration can also be of longer, finite duration. Chronic
administration is administration of a continuous and indefinite
duration, and can be, for example, administration associated with a
chronic condition such as that involving inappropriate
vasoconstriction of the renal vasculature, where chronic
administration is necessary to restore normal renal function.
The methods of the present invention are also applicable to
treating vascular conditions wherein there is no appreciable
decrease or inhibition of endogenous NO. Such vascular conditions
occur, for example, when endothelial cells experience high shear
from rapid movement of red blood cells passing in contact with, or
in close proximity to, endothelial cells, resulting in increased
ET. The methods of the invention restore NO/ET balance. High shear
is associated with, for example, drug therapy and
atherosclerosis.
While the invention has been particularly shown and described with
reference to certain embodiments, it will be understood by those
skilled in the art that various other changes in form and detail
may be made without departing from the spirit and scope of the
invention.
All scientific papers and patents referred to in this specification
are incorporated in totality by reference herein.
Quantitative Study
The study detailed herein quantitatively characterizes the level of
exogenous NO required to restore `normal` vascular function
following the acute and chronic blockade of NO synthase. Referring
to the Figures, and in particular FIG. 1, the
concentration-response relationship with increasing levels of
sodium nitroprusside (SNP) is markedly shifted for both acute and
chronic NO synthase blockade treated rats. The concentration-mean
arterial pressure response curves were divided into two groups,
concentrations that did not and those that did induce a lowering of
mean arterial pressure in the controls. A similar increased
sensitivity to low levels of SNP in the L-NAME treated rats was
demonstrated both in acute and chronic phases of NO synthase
blockade treatment. This level of SNP administration (0.5 to 8
ug/kg per minute) did not significantly decrease mean arterial
pressure in control rats but did result in a marked decrease in
mean arterial pressure.
Referring now to FIG. 2 there may be seen an illustrative example
of this finding: the depressor response to 2 ug/kg per minute in
the NO synthase blockade phase was superimposed over the 2 ug/kg
per minute response in the control period. The mean arterial
pressure lowering induced by 12-32 ug/kg per minute induced a
similar lowering in both control and treated rats (see FIG. 1,
lower panel), indicating a convergence with respect to the
depressor response to SNP.
Referring now to FIG. 3 it may be seen that the cumulative glyceryl
trinitrate concentration-mean arterial pressure response
demonstrated results similar to SNP. The cumulative
concentration-mean arterial pressure response curve was also
shifted leftward in the NO synthase blockade treated rats. Again,
the similar trend of concentrations that do not lower mean arterial
pressure in the controls, almost completely reversed the NO
synthase blockade induced hypertension.
Further studies assessed the impact of different vasoactive systems
on the relationship between nitric oxide and endothelin and, more
specifically, the low dose effects of nitric oxide and the
suppression of local levels of endothelin (ET), which cause
vasodilation and a normalization of vascular tone in focal regions
of the circulation that are, at least in part, NO deficient. In
these studies, L-NAME was administered to increase blood pressure,
and two other vasodilator agents were employed:
isopropylnorepinephrine (INE, a B.sub.1 /B.sub.2 agonist, see FIG.
8) and the .alpha..sub.1 blocker prazosin (see FIG. 9). INE
activates a cAMP mediated signal transduction system, and prazosin
decreases the activity of an IP.sub.3 signal transduction
system.
FIGS. 8 and 9 provide more detailed information on the mechanism
underlying the increased sensitivity of mean arterial pressure to
NO following NO synthase blockade. As can be seen from FIGS. 8 and
9, neither of these two vasodilator agents was able to normalize
the excessive vasoconstriction that follows NO blockade.
Specifically, the two curves on the left-hand side of FIG. 8
clearly demonstrate that the dose response of mean arterial
pressure to INE is identical both before and after nitric oxide
synthase blockade, indicating that the signal mediated by another
vasodilator, INE, is unchanged in the presence or absence of nitric
oxide. It is widely known that the mechanism of action of INE is
via the activation of cAMP mediated signal transduction. In
contrast the curves on the right-hand side of FIG. 8 indicate that
there is an increase in sensitivity of mean arterial pressure to
the NO donor SNP following the blockade of NO synthase.
From a mechanistic standpoint, INE causes vasodilation by the
activation of the cAMP signal transduction system in the
vasculature, whereas SNP causes vasodilation by the activation of
the cGMP signal transduction system or by direct effects of NO (or
an equivalent). The absence of a shift in sensitivity of mean
arterial pressure to INE after NO blockade indicates that the
increased sensitivity to NO donors such as SNP is independent of
the cAMP signal transduction system, and may be directly dependent
on the cGMP signal transduction system.
FIG. 9 illustrates that increased sensitivity to nitric oxide
donors occurs following NO blockade, but does not occur following
treatment with an .alpha..sub.1 receptor antagonist (prazosin). As
prazosin is a second vasodilator agent, similar to INE, these
agents thus present two examples of different vasodilator
mechanisms that do not influence the relationship between nitric
oxide and endothelin, which relationship is in accordance with the
methods of the present invention.
Therefore, to summarize the data presented in FIGS. 8 and 9, these
data present examples demonstrating the relationship between nitric
oxide and endothelin and the effects of a low-dose NO therapy on
normalizing the affected circulation with respect to endothelin and
maintaining and/or restoring vascular tone. These data also
demonstrate that the imbalance in vascular tone that follows NO
synthase blockade is not due to angiotensin II (data not shown),
the sympathetic nervous system, or vasopressin (for example, via
the IP.sub.3 system), but is specific to the cGMP signal
transduction system or direct effects of nitric oxide or its
equivalent. The data further indicate that the addition of an
.alpha..sub.1 receptor antagonist, in contrast to an endothelin
receptor antagonist, does not restore a normal level of vascular
sensitivity. This is in accordance with the invention in that
decreased levels of NO (via L-NAME) result in a marked
up-regulation of endothelin, such as may be explained by an
uncoupling of cGMP signalling.
FIG. 10 is similar in principle to FIG. 2 and provides a further
quantitation of the `microdose` level of exogenous NO that
maintains a normal level of vascular tone following nitric oxide
synthase blockade with L-NAME. The graph of FIG. 10 illustrates
that administering a level of SNP that lowers blood pressure by 8
mmHg completely prevents the development of hypertension following
even maximal NO synthase blockade (which is usually 40 mmHg). Thus,
in accordance with the invention, it can be seen that a microdose
of SNP causes only a minimum level of vasodilation, but completely
prevents the excessive vasoconstriction which occurs following high
level NO synthase blockade using L-NAME.
Further evidence supports that nitric oxide chronically suppresses
the effects of endothelin. As shown in FIG. 11, even normal levels
of NO induce changes in the vasculature by countering the actions
of endothelin. In FIG. 11 (top panel), there can be seen an
increase in the sensitivity of mean arterial pressure to SNP
following NO synthase blockade, and a decrease in the sensitivity
to SNP in the presence of PD145065, an endothelin receptor
antagonist. The rightward shift in the curve for PD145065 (under
control conditions) clearly indicates that the response to NO by
itself has been severely blunted. The bottom panel of FIG. 11
similarly indicates that the increased sensitivity to the NO donor
GTN in response to treatment with L-NAME is abolished in the
presence of PD145065. These results indicate that endothelin
receptors are required to maintain the normal level of nitric oxide
vasodilatory capacity under control conditions.
As would be apparent to a person of ordinary skill in the art, it
is reasonable to use the rat as a model for the affected vascular
systems discussed herein, such as, for example the pudendal and
penis vasculature, and to extend such studies to appropriate
dosages and therapies for other subjects, such as humans. As is
evidenced by Mordenti, "Man versus Beast Pharmacokinetic Scaling in
Mammals", J. Pharm. Sci. 75:1028-1040 (1986) and similar articles,
dosage forms for animals, such as for example rats, can be and are
widely used directly to establish dosage levels in human
applications. One of the present inventors contributed to the
development of a bioassay of erectile function in a rat model at
least as early as 1991 (Heaton et al., J. Urol. 145:1099-1102,
1991), and also helped demonstrate in comparative tests of erectile
function in humans and rats, that the narrow effective dose window
of an orally administered drug, apomorphine, is almost identical
when suitably adjusted for the differences in body weight as taught
by Mordenti, cited above (Heaton et al., Urology 45:200-206,
1995).
Effect of NO in Renal Vasculature
In essence, the kidney functions as a reverse filter in that almost
all of the contents of the blood are filtered into beginning
"urine" of the kidney and the kidney specifically reabsorbs what it
wants to retain later (in the nephrons and loop of Henle). A
physical force, called the renal interstitial hydrostatic pressure
(RIHP), in the tissues of the kidney can have a profound effect on
this process. Increased RIHP presents a physical force opposing the
reabsorption process affecting reabsorption of sodium, water and
other parts of the beginning filtered urine. If not reabsorbed the
result is a diuretic response (natriuresis). In cases of low RIHP
the opposite occurs. Removal of the physical force opposing
reabsorption results in volume accumulation (more blood
volume=higher blood pressure). This pressure-mediated natriuresis
is explained by the fact that some of the blood vessels in the
kidney are not "auto regulated" (much like the penile vasculature),
such that the pressure generated in the kidney tissue (RIHP) is
dictated by the level of vasoconstriction/kidney perfusion.
Excessive vasoconstriction results in a decrease in RIHP, thereby
removing a strong mechanism by which the kidney prevents the
reabsorption of the contents of filtered urine.
It is possible to simulate a type of renal failure, where excessive
vasoconstriction predominates. In these circumstances a volume
conservation situation is created. This simulation is carried out
by treatment of anaesthetized rats which are fitted with
instrumentation for measuring RIHP and mean arterial blood pressure
with NO synthase blockers (L-NAME), The results from such
experiments are depicted in FIGS. 5, 6, and 7 which illustrate
"raw" pressure tracings of an example of these preparations
(Methods written below, see EXPERIMENTAL ASPECTS OF STUDIES
REPORTED: Effects of NO in Renal Vasculature). The traces
illustrate that administration of NO synthase blocker L-NAME,
results in a sharp decline in RIHP (FIG. 5) consequent upon
vasoconstriction of the vasculature. Administration of the NO
precursor L-Arginine (150-200 mg/kg per min) reversed the renal
vasoconstriction (as measured by RIHP changes--see FIG. 6) but did
not alter the level of mean arterial pressure. When the L-Arginine
administration is stopped (with continued administration of L-NAME,
the RIHP once again sharply declines as illustrated in FIG. 7.
Clinical Data: For erectile dysfunction reversal with microdoses of
NO General Methods. Measurements were made in a clinical erectile
dysfunction (ED) laboratory in patients with previously diagnosed
ED. These patients were being evaluated for the purpose of
optimization of their intracernous injection dosing. All men were
assessed using the Queen's University Human Sexuality Group
Protocol (Kingston, Ontario, Canada).
Case 1
The first patient was a man with total erectile dysfunction of 18
months. History of hypertension, myocardial infarct (.times.2),
coronary artery by-pass graft surgery, cerebral vascular accident
and peripheral vascular disease. He was prescribed 0.2 mg/hr nitro
patch (GTN, Ciba-Geigy) with no effect on systemic blood pressure.
The therapy with 0.2 mg/hr nitro patch was on 2 occasions with 100%
successful intercourse. He had angina on the second try. This
result supports the view that the quantity of NO administered was
working at a level significantly lower than quantities typically
used.
Case 2
This patient was a man with no response to intra cavernosal
injection therapy with conventional drugs. 0.2 mg/hr nitro patch
(Ciba-Geigy) followed by 10 mcg PGE1. The patient experienced the
first erection in 4 years (predating radical prostate surgery).
The concentration of glyceryl trinitrate (the typical form of NO
donor derived from Nitrospray or Nitropatches) has been reported in
the literature for such delivery routes to be in the order of 200
to 400 pg/ml of plasma (Sun et al., J. Clin. Pharmacol. 35:390,
1995). Based on the clinical case studies performed, it was noted
that the beneficial effects of a 0.2 mg/h patch are observed after
20 to 30 minutes. This corresponds to a plasma concentration of 100
pg/ml. In respect of the application of the invention to ED therapy
a preferred range of NO agent (such as GTN) is a steady state
plasma concentration of about 50 to about 200 pg/ml. This range
corresponds to the "window" referred to above as illustrated in
FIG. 4.
Support for the proposal that the mechanism of action of the NO
donors delivered at microdoses disclosed herein, are not directly
vasodilatory, can be accomplished by giving animals NOS blocker
high doses to create hypertension. This would be followed by
infusion of an endothelin antagonist at a concentration which would
almost completely reverse the hypertension induced by the NOS
blocker. Subsequently, an NO donor, or combination of NO donors is
infused. The concentration response curve from this infusion should
now be similar to that obtained in control animals. In other words,
the doses of NO which will cause a lowering of pressure will be at
higher values, namely 10 to 24 times higher that in an L-NAME
blocked animal alone thereby showing that high doses of NO are
vasodilatory and lower ones are, on their own, not vasodilatory.
Rather, they act to suppress ET.
While the routes of administration of the NO donors reported here
include intracavernous (IC) injection of NO donor, or by nasal
spray or by patch, the present invention includes administration by
means of topical creams, pharmaceutically acceptable organic and
inorganic carrier substances suitable for parenteral, enteral,
intraurethral, vaginal application, or transmucosal application
via, for example, the respiratory tract (e.g., by inhalation, such
as through intranasal application), which do not deleteriously
react with the active compounds.
Compositions of the invention are administered to subjects in a
biologically compatible form suitable for pharmaceutical
administration in vivo. By "biologically compatible form suitable
for administration in vivo" is meant a form of the active compounds
of the invention to be administered in which any toxic effects are
outweighed by the therapeutic effects of the active compounds of
the invention. The term subject is intended to include living
organisms in which a response can be elicited, e.g., mammals.
Examples of subjects include humans, dogs, cats, mice, rats, and
transgenic species thereof.
Administration of a therapeutically active amount of the
therapeutic compositions of the present invention is defined as an
amount effective, at dosages and for periods of time necessary to
achieve the desired result. For example, a therapeutically active
amount of active compounds of the invention may vary according to
factors such as the disease state, age, sex, and weight of the
individual, and the ability of an agent or combination of agents of
the invention to elicit a desired response in the individual.
Dosage regimens may be adjusted to provide the optimum therapeutic
response. For example, several divided doses may be administered or
the dose may be proportionally reduced as indicated by the
exigencies of the therapeutic situation.
The active compounds (e.g., SNP) may be administered in a
convenient manner such as by injection (subcutaneous, intravenous,
intracavernous, etc.), oral administration, inhalation, transdermal
application, rectal administration, urethral administration,
vaginal administration, or intracavernous introduction. Depending
on the route of administration, the active compound or compounds
may be coated in a material to protect the compound(s) from the
action of enzymes, acids and other natural conditions which may
inactivate the compound(s), or facilitate or enable delivery of
said compound(s).
A SNP composition (or other NO donor) or as a separate agent can be
administered to a subject in an appropriate carrier or diluent,
co-administered with enzyme inhibitors or in an appropriate carrier
such as liposomes. The term "pharmaceutically acceptable carrier"
as used herein is intended to include diluents such as saline and
aqueous buffer solutions and vehicles of solid, liquid or gas
phase. To administer an agent or agents of the present invention by
other than parenteral administration, it may be necessary to coat
the active compound(s) of the invention with, or co-administer the
agent or agents of the present invention with, a material to
prevent its inactivation. Liposomes include water-in-oil-in-water
emulsions as well as conventional liposomes (Strejan et al., J.
Neuroimmunol 7:27 1984). The active compound(s) may also be
administered parenterally or intraperitoneally. Dispersions can
also be prepared in glycerol, liquid polyethylene glycols, and
mixtures thereof in oils and other solutions. Under ordinary
conditions of storage and use, these preparations may contain a
preservative to prevent the growth of microorganisms, stability
enhancers and compounds to preserve physical characteristics that
are needed for appropriate delivery.
Pharmaceutical compositions suitable for injectable use include
sterile aqueous solutions (where water soluble) or dispersions and
sterile powders for the extemporaneous preparation of sterile
injectable solutions or dispersion. In all cases, the composition
must be sterile and must be fluid to the extent that easy
syringability exits. It must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The pharmaceutically acceptable carrier can be a solvent or
dispersion medium containing, for example, water, ethanol, polyol
(for example, glycerol, propylene glycol, and liquid polyethylene
glycol, and the like), and suitable mixtures thereof. The proper
fluidity can be maintained, for example, by the use of a coating
such as lecithin, by the maintenance of the required particle size
in the case of dispersion and by the use of surfactants. Prevention
of the action of microorganisms can be achieved by various
antibacterial and antifungal agents, for example, parabens,
chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In
many cases, it will be preferable to include isotonic agents, for
example, sugars, polyalcohols such as mannitol, and sorbitol, or
sodium chloride in the composition. Prolonged absorption of the
injectable compositions can be brought about by including in the
composition an agent which delays absorption, for example, aluminum
monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating
active compound (e.g., SNP) or compounds in the required amount in
an appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by filter sterilization.
Generally, dispersions are prepared by incorporating the active
compound into a sterile vehicle which contains a basic dispersion
medium and the required other ingredients from those enumerated
above. In the case of sterile powders for the preparation of
sterile injectable solutions, the preferred methods of preparation
are vacuum drying and freeze-drying which yields a powder of an
active ingredient of the invention plus any additional desired
ingredient from a previously sterile-filtered solution thereof.
When an active compound(s) is suitably protected, as described
above, the composition may be orally administered, for example,
with an inert diluent or an assimilable edible carrier. As used
herein "pharmaceutically acceptable carrier" includes any and all
solvents, dispersion media, coatings, antibacterial and antifungal
agents, isotonic and absorption delaying agents, and the like. The
use of such medica and agents for pharmaceutically active
substances is well known in the art. Except insofar as any
conventional media or agent is incompatible with the active
compound, use thereof in the therapeutic compositions is
contemplated. Supplementary active compounds can also be
incorporated into the compositions.
It is especially advantageous to formulate parenteral compositions
in dosage unit form for ease of administration and uniformity of
dosage. Dosage unit form as used herein refers to physically
discrete units suited as unitary dosages for mammalian subjects to
be treated; each unit containing a predetermined quantity of active
compound calculated to produce the desired therapeutic effect in
association with the required pharmaceutical carrier. The
specification for the dosage unit forms of the invention are
dictated by and directly dependent on (a) the unique
characteristics of the active compound and the particular
therapeutic effect to be achieved, and (b) the limitations inherent
in the art of compounding such an active compound for the
therapeutic treatment of individuals.
Experimental Aspects of Studies Reported
Quantitative Study
Animals
Male Sprague-Dawley rats (325 to 400 g) obtained from Charles River
Laboratories (Montreal, Canada) were housed individually under
conditions of 12-hour light/dark cycle, with room temperature at 22
to 24.degree. C., and were provided with Purina rodent chow and tap
water ad libitum for at least 2 days before any experiments were
started.
Measurement of MAP and Short Acting Drug Administration
The surgical method was based on the technique of Thompson et al.
(Hypertens. 20:809, 1992). In brief, rats were anaesthetized with
ketamine/xylazine (70/5 mg/kg i.p.), and the descending aorta
distal to the kidneys was catheterized with small bore Teflon
tubing (0.012 in i.d., 30 gauge, Cole-Parmer, Laval, Quebec,
Canada) inserted into vinyl tubing (0.02 in i.d., 0.060 in, 23
gauge). The inferior vena cava was also catheterized distal to the
kidneys with small bore Teflon tubing (0.012 in i.d., 30 gauge,
Cole-Parmer). The catheters were filled with heparinized saline (10
IU/ml) and held in place by a small amount of cyanoacrylate glue at
the puncture site. The catheters were tunnelled subcutaneously and
exteriorized at the back of the neck and sutured in place. Two days
after surgery, MAP could be recorded (MacLab DAS, ADInstruments,
Milford, Mass.). After connection, an equilibration period of
approximately 30 minutes allowed for the determination of the
steady state level of MAP before any recording began. Baseline MAP
was determined from readings averaged over 5 minutes, taken from
each rat at 15 minute intervals for at least 1 hour prior to the
start of any experiment.
Sodium Nitroprusside and Glyceryl Trinitrate Concentration--Mean
Arterial Pressure Response Curves Following Acute and Chronic
N.sup..omega. -nitro-L-arginine Methyl Ester
Rats were randomly assigned treatment with N.sup..omega.
-nitro-L-arginine methyl ester (L-NAME) for 30 minutes (100 mg/kg,
intraperitoneally) or 12 days (100 mg/kg, in drinking water) or tap
water. Two days prior to the day of the experiment rats were
instrumented with catheters, as described above. Following baseline
measurements of MAP, rats were given a infusions of sodium
nitroprusside (SNP, 0.5 to 32 ug/kg per minute dissolved in 0.9%
sterile saline) with a step-wise increase in concentration every
two minutes. Rats were allowed 30 minutes to recover from the SNP
administration. Rats were then given infusions of glyceryl
trinitrate (GTN, 0.5 to 32 ug/kg per minute dissolved in 0.9%
sterile saline) with a step-wise increase in concentration every
two minutes.
Throughout all of these pharmacological manipulations, MAP and HR
were recorded at a sampling rate of 100 Hz and the data was stored
on a disk drive for later analysis.
Effects of NO in Renal Vasculature
Surgical preparation: Experiments were performed in male
Sprague-Dawley rates, 250 to 330 g, obtained from Charles River
Laboratories (Montreal, Canada). Food (Purina rat chow) and water
were provided ad libitum throughout the study. Rats were
acclimatized for at least a week prior to experimentation in group
housing under conditions of a 12-hour light/dark cycle, with room
temperature at 22 to 24.degree. C. 3 to 4 days prior to each
experimental day, rats were instrumented with an aortic catheter as
described in detail previously (Thompson et al., Hypertens.20:809,
1992). In brief, the rates were anaesthetized with ketamine
hydrochloride (70 mg/kg i.p. (Rogarsetic, Rogar/STB Inc., Montreal,
PQ) and xylazine hydrochloride (5 mg/kg i.p. (Rompum, Haver Inc.,
Etobickoe, ON) and placed on a heating pad to maintain a constant
body temperature of 37.degree. C. Additional ketamine was given, as
necessary, during surgery. Following an abdominal incision, the
descending aorta distal to the kidneys was catheterized with
smallbore Teflon tubing (i.d.=0.012 in, o.d.=0.030 in, 30 gauge,
Cole-Parmer, Laval, Quebec, Canada), inserted into vinyl tubing
(i.d.=0.02 in, o.d.=0.060 in, 23 gauge). The catheter was filled
with heparinized saline (50 IU/ml, Grade 1-A Heparin Sodium salt,
Sigma Chemical Co., St Louis) and held in place by a small amount
of cyanoacrylate tissue glue (Lepage Ltd., Brampton, ON). The
catheter was then flushed with a small volume (0.1-0.3 ml) of
heparinized saline (50 IU/ml) and left entirely inside the
abdominal cavity until the day of the RIHP measurements. The
abdominal incision was sutured with 6-0 silk braided thread
(Ethicon Ltd., Peterborough, Ontario). The rats were allowed to
recover for 2 to 5 days, during which they were housed individually
under the same conditions as described above. Rats were re-weighed
following the recovery period and rats that had lost >30 g were
considered to have not sufficiently recovered from the surgery and
hence, were excluded from the study (Haskins, S. C., Postoperative
care, In: Methods of Animal Experimentation, Vol. III Part A, eds.
Gay et al. (Academic Press Inc., Orlando, 1986)).
Experimental Procedure: The RIHP measurements were carried out in
rats anaesthetized with thiobutabarbital sodium (Inactin, 100 mg/kg
i.p., Byk-Gulden, Constance, Germany). This anaesthetic is used
most frequently by others for the study of RIHP and PN and, in
preliminary studies, yielded similar results to those studies
compared to a Ketamine/Xylazine anaesthetic preparation.
Rats were placed on a heating pad to maintain a constant body
temperature of 37.degree. C. and the abdominal incision was
reopened. Hematocrits were determined by the microcapillary tube
method from arterial blood samples (300 .mu.l) obtained from the
arterial catheter. Only rates in which the hematocrit was found to
be between 40 and 45 percent were subsequently used. Rates with
hematocrits out of this range were considered to be hemodynamically
challenged due to the blood loss during surgery and, since
reductions in hematocrit can compromise cardiovascular function,
were not used in this study (Haskins, S. C., Postoperative care,
In: Methods of Animal Experimentation, Vol. III Part A , eds. Gay
et al. (Academic Press Inc., Orlando, 1986); Pirkle et al.,
Endocrin. 110:7, 1982; Houttuin et al., Am J. Physiol. 223:63,
1972).
The arterial catheter, inserted previously, was attached to a
pressure transducer (model CDX3, Cobe), and the pulsatile signal
recorded using a physiograph (Beckman, model R511 or MacLab,
ADInstruments Pty Ltd., Castle Hill, Australia). Small sections of
the aorta (<0.5 cm proximal to the right kidney), mesenteric and
celiac arteries were isolated and silk ligatures were placed
loosely around the vessels.
RIHP was measured by implanting a cannula into the lateral side of
the left renal cortex (Garcia-Estan et al., Am. J. Physiol.,
256:F63, 1989). The RIHP catheter was constructed by inserting a
small core of porous polyethylene matrix material (2 mm, 35 .mu.m
pore size, Bel-Art Products, Pequannoc, N.J.) into the heat
expanded end of a polyethylene catheter (PE-50, i.d.=0.023 in,
o.d.=0.038 in.times.10-20 cm) such that approximately one-third of
the matrix extends beyond the tubing. This technique ensures that
tissue does not block the catheter while allowing for measurement
of hydrostatic pressure (Roman et al., Am. J. Physiol. 248:F190,
1985; Ott et al., J. Appl. Physiol. 38:937, 1975). A 3 mm deep hole
was made in the lateral surface of the left kidney with an
electrocautery needle (26 gauge) and current was passed through the
needle for approximately 3 to 5 seconds. Bleeding was completely
stopped by applying soft pressure with a cotton swab. The kidney
was kept dry before implanting the RIHP catheter. The RIHP catheter
was inserted into the hole and then sealed to the surface of the
kidney capsule with cyanoacrylate. The RIHP catheter was similarly
attached to the physiograph via the pressure transducer. Lack of
pulsatile pressure ensured that the catheter was not in a blood
vessel. To further check the location of the catheter two
procedures were performed: (i) a small volume (100-200 .mu.l) of
saline (10 IU/ml heparin) was slowly infused in order to obtain a
characteristic increase in RIHP (Ott et al., 1975) and (ii) a RIHP
response to renal vein occlusion (RVO) was observed (Garcia-Estan
et al., Am. J. Physiol., 256:F63, 1989). Rats that failed to show
these characteristic responses (FIG. 2-1) were excluded from the
experiment
After a 10 to 15 minute equilibration period, the RVO response from
the RIHP catheter and a pulsatile recording from the arterial
catheter were re-confirmed and a baseline MAP and RIHP were
recorded. During the control periods of the experiment, the exposed
abdominal cavity was kept moist by covering with a wet gauze patch
and 0.9% saline. In each experiment, arterial pressure was
manipulated systematically from low to high MAP by sequentially
tightening the ligatures around the upper aorta, celiac artery and
mesenteric artery. Pressure changes were initially maintained for
approximately 5 to 7 minutes in order to ensure that both MAP and
RIHP equilibrate to the new steady state. As it was determined that
RIHP equilibrated in less than 3 minutes all date was collected
within this time. The RVO-RIHP response was checked periodically
after reaching different arterial pressures. This sequence of
arterial pressure manipulations was repeated after a 10 to 15
minute control period at baseline MAP and RIHP. The position of the
RIHP catheter was verified at the end of each experiment and was
typically found in the corticomedullary junction.
Those skilled in the art will recognize, or be able to ascertain
through routine experimentation, equivalents to the specific
embodiments described herein. Such equivalents are considered to be
within the scope of the invention and are covered by the appended
claims.
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